Functional analysis of glyco-molecules that bind with influenza virus.

Some animal influenza A viruses (IAVs) bind not only to N-acetylneuraminic acid (Neu5Ac) but also to N-glycolylneuraminic acid (Neu5Gc), which has been discussed as a virus receptor. Human cells cannot synthesize Neu5Gc due to dysfunction of the CMP-Neu5Ac hydroxylase (CMAH) gene, which converts CMP-Neu5Ac to CMP-Neu5Gc. However, exogenous Neu5Gc from Neu5Gc-rich dietary sources is able to be metabolically incorporated into surfaces of tissue cells and may be related to enhancement of the infectivity and severity of IAV. Here, we investigated the receptor function of Neu5Gc on IAV infection in Neu5Gc-expressing cells by transfection of the monkey CMAH gene into human cells or by incubation with human cells in the presence of N-glycolylmannosamine. Expression of Neu5Gc on human cells clearly suppressed infectivity of IAVs that possess Neu5Gc binding ability. Furthermore, there was no difference in infectivity of a transfectant virus that included the wild-type HA gene from A/Memphis/1/1971 (H3N2), which shows no Neu5Gc binding, between parent MCF7 cells and cells stably expressing the monkey CMAH gene (CMAH-MCF7 cells). On the other hand, cell entry of the transfectant virus that included the Neu5Gc-binding HA gene with a single mutation to Tyr at position Thr155 was arrested at the stage of internalization from the plasma membrane of the CMAH-MCF7 cells. These results indicate that expression of Neu5Gc on the surface of human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to work as a decoy receptor of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. Influenza A viruses (IAVs) bind to the host cell surfaces through sialic acids at the terminal of glycoconjugates. For IAV binding to sialic acids, some IAVs bind not only to N-acetylneuraminic acid (Neu5Ac) as a receptor but also to N-glycolylneuraminic acid (Neu5Gc). Neu5Gc has been discussed as a receptor of human and animal IAVs. Our results showed that Neu5Gc expression on human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to be a "decoy receptor" of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. Human cells cannot synthesize Neu5Gc because of dysfunction of the CMP-N-acetylneuraminic acid hydroxylase gene but can exogenously and metabolically incorporate Neu5Gc from dietary sources. The expression of Neu5Gc on human epithelial cells by taking in exogenous Neu5Gc from Neu5Gc-rich dietary sources may be related to restriction of the infection of IAVs that have acquired Neu5Gc binding ability.

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Influenza A viruses (IAVs) can jump species barriers and occasionally cause epidemics, epizootics, pandemics, and panzootics. Characterizing the infection dynamics at the target tissues of natural hosts is central to understanding the mechanisms that control host range, tropism, and virulence. Canine influenza virus (CIV; H3N8) originated after the transfer of an equine influenza virus (EIV) into dogs. Thus, comparing CIV and EIV isolates provides an opportunity to study the determinants of influenza virus emergence. Here we characterize the replication of canine, equine, and human IAVs in the trachea of the dog, a species to which humans are heavily exposed. We define a phenotype of infection for CIV, which is characterized by high levels of virus replication and extensive tissue damage. CIV was compared to evolutionarily distinct EIVs, and the early EIV isolates showed an impaired ability to infect dog tracheas, while EIVs that circulated near the time of CIV emergence exhibited a CIV-like infection phenotype. Inoculating dog tracheas with various human IAVs (hIAVs) showed that they infected the tracheal epithelium with various efficiencies depending on the virus tested. Finally, we show that reassortant viruses carrying gene segments of CIV and hIAV are viable and that addition of the hemagglutinin (HA) and neuraminidase (NA) of CIV to the 2009 human pandemic virus results in a virus that replicates at high levels and causes significant lesions. This provides important insights into the role of evolution on viral emergence and on the role of HA and NA as determinants of pathogenicity. Influenza A viruses (IAVs) have entered new host species in recent history, sometimes with devastating consequences. Canine influenza virus (CIV) H3N8 originated from a direct transfer of an equine influenza virus (EIV) in the early 2000s. We studied the infection patterns of IAVs that circulate in dogs or to which dogs are commonly exposed and showed that CIV emergence was likely caused by an adaptive driver, as evolutionarily distinct EIVs display distinct infection phenotypes. We also showed that many human viruses can infect dog tracheas and that reassortment with CIV results in viable viruses. Finally, we showed that the hemagglutinin and neuraminidase of CIV act as virulence factors. Our findings have significant implications because they show that dogs might act as "mixing vessels" in which novel viruses with pandemic potential could emerge and also provide experimental evidence supporting the role of viral evolution in influenza virus emergence.

Pigs play an important role in the interspecies transmission of influenza A viruses (IAV). The porcine airway epithelium contains binding sites for both swine/human IAV (α2,6-linked sialic acids) and avian IAV (α2,3-linked sialic acids) and therefore is suited for adaptation of viruses from other species as suggested by the “mixing vessel theory”. Here, we applied well-differentiated swine airway epithelial cells to find out whether efficient infection by avian IAV requires prior adaption. Furthermore, we analyzed the influence of the sialic acid-binding activity and the virus-induced detrimental effects. Surprisingly, an avian IAV H1N1 strain circulating in European poultry and waterfowl shows increased and prolonged viral replication without inducing a strong innate immune response. This virus could infect the lower respiratory tract in our precision cut-lung slice model. Pretreating the cells with poly (I:C) and/or JAK/STAT pathway inhibitors revealed that the interferon-stimulated innate immune response influences the replication of avian IAV in swine airway epitheliums but not that of swine IAV. Further studies indicated that in the infection by IAVs, the binding affinity of sialic acid is not the sole factor affecting the virus infectivity for swine or human airway epithelial cells, whereas it may be crucial in well-differentiated ferret tracheal epithelial cells. Taken together, our results suggest that the role of pigs being the vessel of interspecies transmission should be reconsidered, and the potential of avian H1N1 viruses to infect mammals needs to be characterized in more detail.

Alveolar macrophages (AM) play pivotal roles in modulating host defense, pulmonary inflammation, and tissue injury following respiratory viral infections. However, the transcriptional regulation of AM function during respiratory viral infections is still largely undefined. Here we have screened the expression of 84 transcription factors in AM in response to influenza A virus (IAV) infection. We found that the transcription factor PPAR-γ was downregulated following IAV infection in AM through type I interferon (IFN)-dependent signaling. PPAR-γ expression in AM was critical for the suppression of exaggerated antiviral and inflammatory responses of AM following IAV and respiratory syncytial virus (RSV) infections. Myeloid PPAR-γ deficiency resulted in enhanced host morbidity and increased pulmonary inflammation following both IAV and RSV infections, suggesting that macrophage PPAR-γ is vital for restricting severe host disease development. Using approaches to selectively deplete recruiting monocytes, we demonstrate that PPAR-γ expression in resident AM is likely important in regulating host disease development. Furthermore, we show that PPAR-γ was critical for the expression of wound healing genes in AM. As such, myeloid PPAR-γ deficiency resulted in impaired inflammation resolution and defective tissue repair following IAV infection. Our data suggest a critical role of PPAR-γ expression in lung macrophages in the modulation of pulmonary inflammation, the development of acute host diseases, and the proper restoration of tissue homeostasis following respiratory viral infections.IMPORTANCE Respiratory viral infections, like IAV and respiratory syncytial virus (RSV) infections, impose great challenges to public health. Alveolar macrophages (AM) are lung-resident immune cells that play important roles in protecting the host against IAV and RSV infections. However, the underlying molecular mechanisms by which AM modulate host inflammation, disease development, and tissue recovery are not very well understood. Here we identify that PPAR-γ expression in AM is crucial to suppress pulmonary inflammation and diseases and to promote fast host recovery from IAV and RSV infections. Our data suggest that targeting macrophage PPAR-γ may be a promising therapeutic option in the future to suppress acute inflammation and simultaneously promote recovery from severe diseases associated with respiratory viral infections.

Between 2013 and 2018, the novel A/Anhui/1/2013 (AH/13)-lineage H7N9 virus caused at least five waves of outbreaks in humans, totaling 1,567 confirmed human cases in China. Surveillance data indicated a disproportionate distribution of poultry infected with this AH/13-lineage virus, and laboratory experiments demonstrated that this virus can efficiently spread among chickens but not among Pekin ducks. The underlying mechanism of this selective transmission remains unclear. In this study, we demonstrated the absence of Neu5Gc expression in chickens across all respiratory and gastrointestinal tissues. However, Neu5Gc expression varied among different duck species and even within the tissues of the same species. The AH/13-lineage viruses exclusively bind to acetylneuraminic acid (Neu5Ac), in contrast to wild waterbird H7 viruses that bind both Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The level of Neu5Gc expression influences H7 virus replication and facilitates adaptive mutations in these viruses. In summary, our findings highlight the critical role of Neu5Gc in affecting the host range and interspecies transmission dynamics of H7 viruses among avian species.IMPORTANCEMigratory waterfowl, gulls, and shorebirds are natural reservoirs for influenza A viruses (IAVs) that can occasionally spill over to domestic poultry, and ultimately humans. This study showed wild-type H7 IAVs from waterbirds initially bind to glycan receptors terminated with N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). However, after enzootic transmission in chickens, the viruses exclusively bind to Neu5Ac. The absence of Neu5Gc expression in gallinaceous poultry, particularly chickens, exerts selective pressure, shaping IAV populations, and promoting the acquisition of adaptive amino acid substitutions in the hemagglutinin protein. This results in the loss of Neu5Gc binding and an increase in virus transmissibility in gallinaceous poultry, particularly chickens. Consequently, the transmission capability of these poultry-adapted H7 IAVs in wild water birds decreases. Timely intervention, such as stamping out, may help reduce virus adaptation to domestic chicken populations and lower the risk of enzootic outbreaks, including those caused by IAVs exhibiting high pathogenicity.

Calcium (Ca2+) dependent signaling circuit plays a critical role in influenza A virus (IAV) infection. The 8-O-(E-p-methoxycinnamoyl)harpagide (MCH) exhibits pharmacological activities that exert neuroprotective, hepatoprotective, anti-inflammatory and other biological effects. However, not have reports of antiviral effects. To investigate the antiviral activity of MCH on IAV-infected human lung cells mediated by calcium regulation. We examined the inhibitory effect of MCH on IAV infections and measured the level of viral proteins upon MCH treatment using Western blotting. We also performed molecular docking simulation with MCH and IAV M2 protein. Finally, we analyzed MCH’s suppression of intracellular calcium and ROS (reactive oxygen species) in IAV-infected human lung cells using a flow cytometer. The results shown that MCH inhibited the infection of IAV and increased the survival of the infected human lung cells. The levels of IAV protein M1, M2, NS1 and PA were inhibited in MCH-treated human lung cells compared to that in infected and untreated cells. Also, docking simulation suggest that MCH interacted with M2 on its hydrophobic wall (L40 and I42) and polar amino acids (D44 and R45), which formed intermolecular contacts and were a crucial part of the channel gate along with W41. Lastly, MCH inhibited IAV infection by reducing intracellular calcium and mitochondrial Ca2+/ROS levels in infected human lung cells. Taken together, these data suggest that MCH inhibits IAV infection and increases the survival of infected human lung cells by suppressing calcium levels. These results indicate that MCH is useful for developing IAV treatments.

Wild birds of Anseriformes and Charadriiformes are natural reservoirs of influenza A viruses (IAVs). Occasionally, IAVs transmit and adapt to mammalian hosts, and are maintained as epidemic strains in their new hosts. Viral adaptions to mammalian hosts include altered receptor preference of host epithelial sialylated oligosaccharides from terminal α2,3-linked sialic acid (SA) towards α2,6-linked SA. However, α2,3-linked SA has been found in human respiratory tract epithelium, and human infections by avian IAVs (AIVs) have been reported. To further explore the attachment properties of AIVs, four AIVs of different subtypes were investigated on human and pig tissues using virus histochemistry. Additionally, glycan array analysis was performed for further characterization of IAVs’ receptor structure tropism. Generally, AIV attachment was more abundant to human tissues than to pig tissues. The attachment pattern was very strong to human conjunctiva and upper respiratory tract, but variable to the lower respiratory tract. AIVs mainly attached to α2,3-linked SA, but also to combinations of α2,3- and α2,6-linked SA. The low attachment of these AIV isolates to pig tissues, but high attachment to human tissues, addresses the question whether AIVs in general require passage through pigs to obtain adaptions towards mammalian receptor structures.

Influenza A virus (IAV) is a major public health problem responsible for the death of half
\na million people every year worldwide. Zoonotic transmissions of the virus from swine and avian
\norigin have occurred and can in the worst-case result in pandemics. Prediction of the next
\npandemic strain is still a major challenge as mechanisms of antigenic shift and the zoonotic
\nnature of influenza viruses are still poorly understood. On the other hand, the current vaccination
\nstrategy treating seasonal influenza viruses, given its own problems in efficacy, is not well suited
\nto mitigate an impending pandemic. While there are few clinically approved anti-IAV drugs for
\ntherapeutic and prophylactic use, these drugs have also been challenged by the emergence of
\ndrug resistance, toxicity, adverse effects and low efficacy. Hence, development of a broad range
\nanti-IAV drug that could target all of the IAV strains irrespective of their source is critically
\nimportant.
\nTo discover effective IAV vaccines and alternative anti-IAV therapeutics, it is crucial to
\nunderstand how IAVs adapt to different species’ host ranges and vice versa, how different
\nspecies hosts respond to different IAV subtypes. Additionally, it is crucial to understand IAVhost interactions and identifying critical viral or host factors that could support the replication of
\nthe virus or induce pathologic conditions. Recently, high throughput technologies such as mRNA
\nmicroarray-based gene expression, genome-wide siRNA screening, and proteomic analysis are
\nproviding in-depth insights into host-pathogen interactions of IAVs. Hence, this study aimed to
\ncomputationally investigate IAV-host interactions using three data set (transcriptome, genomewide siRNA screens and interactome) and identify 1) virus and host specific responses, 2) host
\ndeterminants in host adaptations and 3) host targets for IAV therapeutics.
\nThis thesis contains four main projects. In the first project (chapter 3), we investigated the
\nhost gene expression changes of eight IAVs (H1N1/WSN, pH1N1, H5N2/F118, H5N2/F59,
\nH5N2/F189, H5N3, H7N9 and H9N2 viruses) in A549 cells at different time points of infections.
\nThen we integrated the differentially expressed genes (DEGs) in at least 3 viruses, with 1713 and
\n1780 host factors comprehensively curated from 11 siRNA and 13 interactome studies
\nrespectively. The integration of the three data set highlighted plausible influenza A virus required
\nhost factors (IHFs) that could be targeted against IAVs. The up-regulated IHFs (e.g. TRIM21,
\nTRIM26, IRF2, and SAMHD1) might support the replication of IAV through suppression of the innate and adaptive antiviral immune responses. The other up-regulated IHFs could also enhance
\nthe replication of IAV at different stages of the virus lifecycle: endocytosis (BTC), prevention of
\napoptosis (TIMM17A), nuclear import (JAK2), and translation elongation of viral proteins
\n(HEXIM1). Although several of these IHFs have been implicated in other viruses, the detailed
\nmechanisms of how several of these up-regulated IHFs could support IAV replication require
\nfurther investigation.
\nThe second project (chapter 4), explored a comprehensive analysis of host gene
\nexpression changes in different IAV infections in different host species. We used host gene
\nexpression signatures of cell lines from three species (A549 (human), CEF (chicken), and
\nMDCK (canine) in response to six IAVs (H1N1/WSN, H5N2/F59, H5N2/F118, H5N2/F189,
\nH5N3 and H9N2 viruses). To compare the expression changes between the three species, we
\nperformed comprehensive probe set re-annotation and human ortholog mapping. The result
\nshowed that the expression signatures of different IAV isolates in a single species cell type are
\nmore similar to each other compared to the expression signatures of a single isolate in different
\nspecies’ cell types. The functional annotations (pathways) and the highly expressed cell-specific
\nsignatures indicated that IAVs up-regulated host factors could induce virus infectivity (e.g.
\nOSBPL1A and ARHGAP21), reduce apoptosis (e.g. MRPS27) and increase cell proliferation (e.g.
\nCOPS2) in CEF cells. Conversely, increased antiviral, pro-apoptotic and inflammatory
\nsignatures have been identified in A549 cells. Except in H5N3 virus infections, generally IAV
\ninfections down-regulates genes associated with cell cycle and metabolic pathways with the
\nstrongest effect in MDCK cells, followed by A549 cells in a strain dependent manner, but not in
\nCEF cells. Previously our group demonstrated that the replication of the examined viruses was
\nsignificantly higher in CEF cells than the other cell types. Thus, we hypothesise that this could
\npartially explain the mechanism how infectious LPAI viruses shed by chickens lack the
\ninflammatory response and cellular disruption that may lead to disease conditions.
\nIn the third project (chapter 5), we used a systems-based approach to investigate changes
\nto the transcriptome of primary murine lung macrophages (PMФ) in response to infection with
\nthe mouse-adapted H1N1/WSN virus and low pathogenic avian influenza (LPAI) viruses H5N2
\nand H5N3. The results showed that while all viruses induced antiviral responses, the H5N3
\ninfection resulted in higher expression levels of cytokines and chemokines associated with
\ninflammatory responses. Previously, our group showed that the LPAI H5N2 and H5N3 were able to infect murine lung macrophages and together with increased expression of inflammatory
\nmediators particularly in H5N3 virus could impose threats to human health in the future.
\nThe IHFs identified by the IAV genome-wide siRNA screening studies could be used as
\npotential anti-IV targets. However, these studies were not consistent (reproducible) mainly due to
\nfalse negative results. Hence, in the fourth project (chapter 6), we applied computational gene
\nnetwork growing for discovering gene network links that could have been missed by these
\nexperimental investigations. Using the known IHFs we compared the network growing function
\nof two free tools GeneMANIA and STRING and the commercial IPA for their performance of
\nrecovering other known IHFs previously identified from siRNA screens. The result showed that,
\ngiven small (~30 genes) or medium (~150 genes) input sets, all three network growing tools
\ndetected significantly more known host factors than random human genes with STRING overall
\nperforming strongest. Extending the networks with all the three tools significantly improved the
\ndetection of GO biological processes of known host factors compared to not growing networks.
\nNotably, the rate of identification of true host factors using computational network growing is
\nequal or better to doing another experimental siRNA screening study which could also be true
\nand applied to other biological pathways/processes.
\nFinally, using the known IHFs and the "new IHF candidates" (genes connected to the
\nIHFs from the network growing analysis), we predicted computational drug-target interactions
\nusing MetaCore. We identified 343 US Food and Drug Administration (FDA) approved drugs
\nthat had an inhibitory effect on either the known or new candidate IHFs, of which 258 were new
\npredictions. Furthermore, using different criteria, we computationally ranked the 343 FDA
\napproved drugs for further experimental validation.

Influenza A viruses (IAVs) rely on viral ribonucleoprotein (vRNP) complexes to control transcription and replication. Each vRNP consists of one viral genomic RNA segment associated with multiple nucleoproteins (NP) and a trimeric IAV RNA polymerase complex. Previous studies showed that post-translational modifications of vRNP components, such as NP, by host factors would in turn affect the IAV life cycle or modulate host anti-viral response. In this study, we found host E3 ubiquitin ligase Pirh2 interacted with NP and mediated short-chain ubiquitination of NP at lysine 351, which suppressed NP-PB2 interaction and vRNP formation. In addition, we showed that knockdown of Pirh2 promoted IAV replication, whereas overexpression of Pirh2 inhibited IAV replication. However, Pirh2-ΔRING lacking E3 ligase activity failed to inhibit IAV infection. Moreover, we showed that Pirh2 had no effect on the replication of a rescued virus, WSN-K351R, carrying lysine-to-arginine substitution at residue 351. Interestingly, by analyzing human and avian IAVs from 2011 to 2020 in influenza research databases, we found that 99.18% of 26 977 human IAVs encode lysine, but 95.3% of 9956 avian IAVs encode arginine at residue 351 of NP protein. Consistently, knockdown of Pirh2 failed to promote propagation of two avian-like influenza viruses, H9N2-W1 and H9N2-C1, which naturally encode arginine at residue 351 of NP. Taken together, we demonstrated that Pirh2 is a host factor restricting IAV infection by modulating short-chain ubiquitination of NP. Meanwhile, it is noteworthy that residue 351 of NP targeted by Pirh2 may associate with the evasion of human anti-viral response against avian-like influenza viruses.

Influenza A viruses (IAVs) continue to threaten animal and human health globally. Bats are asymptomatic reservoirs for many zoonotic viruses. Recent reports of two novel IAVs in fruit bats and serological evidence of avian influenza virus (AIV) H9 infection in frugivorous bats raise questions about the role of bats in IAV epidemiology. IAVs bind to sialic acid (SA) receptors on host cells, and it is widely believed that hosts expressing both SA α2,3-Gal and SA α2,6-Gal receptors could facilitate genetic reassortment of avian and human IAVs. We found abundant co-expression of both avian (SA α2,3-Gal) and human (SA α2,6-Gal) type SA receptors in little brown bats (LBBs) that were compatible with avian and human IAV binding. This first ever study of IAV receptors in a bat species suggest that LBBs, a widely-distributed bat species in North America, could potentially be co-infected with avian and human IAVs, facilitating the emergence of zoonotic strains.

SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase protein 1 (SAMHD1) is an intracellular protein that regulates a stable deoxynucleoside triphosphates (dNTPs) for normal cellular function. Many studies have documented SAMHD1-mediated restriction of RNA retroviruses and different DNA viruses in non-dividing myeloid cells, where it acts to deplete cellular dNTP pools to inhibit replication of the viral genome. Less is currently known regarding its ability to restrict the replication of other RNA viruses in different cell types. In this study, we investigate the role of SAMHD1 in modulating the replication of RNA viruses associated with respiratory virus infection, with a particular focus on seasonal influenza A virus (IAV). Moreover, our studies have assessed the ability of SAMHD1 to modulate IAV replication in two key cell types associated with the pathogenesis of IAV, namely epithelial and macrophage-like cells. qPCR was used to examine expression of endogenous SAMHD1 in response to IAV infection and/or treatment with recombinant type I interferon (IFN) in epithelial (A549) and macrophage-like (THP-1) cells. CRISPR/Cas9 knockout (KO) of endogenous SAMHD1 was performed to assess the role of SAMHD1 in modulating IAV replication in A549 cells or in differentiated (d)THP-1. IAV infection and replication were also assessed in A549 and dTHP-1 SAMHD1 KO cells with stable doxycycline (DOX)-induced expression of SAMHD1 or of phosphorylation mutants of SAMHD1. CRISPR/Cas9 KO of endogenous SAMHD1 in dTHP-1 or A549 cells did not enhance virus release following infection with seasonal IAV. Moreover, reconstitution of KO cells with DOX-inducible SAMHD1 did not restrict the growth of seasonal IAV in dTHP-1 or A549 cells, despite SAMHD1-mediated restriction of a DNA virus (HSV-1) in both cell types. In contrast to seasonal IAV, we also observed restriction of highly pathogenic avian influenza (HPAI) A(H5N1) IAV replication in dTHP-1 cells expressing DOX-inducible SAMHD1. Together, these studies indicate that neither endogenous nor DOX-inducible SAMHD1 restricts the growth of seasonal IAV in macrophage-like or epithelial cells.

Evaluation of dam parity and internal biosecurity practices in influenza infections in piglets prior to weaning

Rationale: Influenza A virus (IAV) poses a major public health threat, causing severe lung inflammation. Due to frequent mutations, IAVs often develop resistance to current antiviral treatments. Hence, to identify potential alternative therapies, it is crucial to understand how IAVs interact with host proteins. Our previous research showed that IAV induces the host cellular protein Bcl-2 interacting killer (BIK), promoting viral replication in airway epithelial cells (AECs). However, the precise mechanism remains unclear. Here, we further investigated how IAV hijacks the host cellular proteins to promote viral replication and exacerbate lung inflammation. Methods: Human precision-cut lung slices (hPCLS) were transduced with adeno-associated viral (AAV) vectors expressing EGFP or ARIH2, followed by IAV infection. To explore the translational potential, ARIH2 was delivered intranasally via AAV6.2 vector in C57BL/6 mice followed by IAV-infection, and the impact on lung inflammation and survival was evaluated. Lung histology was assessed using hematoxylin and eosin (H&E) staining. Western blot, co-immunoprecipitation, ubiquitination assays, mass spectrometry (MS), and immunofluorescence were performed to assess protein expression. Luminex multiplex assays quantified inflammatory cytokines/chemokines in lung homogenates and supernatants of IAV-infected mice and hPCLS. Results: MS analysis identified ARIH2 as the primary BIK-interacting E3 ligase suppressed by IAV to stabilize BIK. This finding was supported by the observation that decreasing ARIH2 levels increased BIK protein levels in IAV-infected AECs. IAV nucleoprotein, distinct from other viral ribonucleoprotein components, specifically inhibited ARIH2 and induced BIK. Overexpression of ARIH2 reduced BIK levels and diminished IAV yield. Conversely, ARIH2 depletion led to increased BIK protein levels. Furthermore, ARIH2 showed a robust interaction with BIK. IAV inhibited ARIH2-mediated K48-linked BIK ubiquitination. On the contrary, ARIH2 overexpression in IAV-infected cells restored BIK ubiquitination and dampened IAV-induced pro-inflammatory cytokines, affirming that IAV interferes with ARIH2-mediated BIK degradation. In hPCLS, IAV infection suppressed ARIH2, thereby increasing BIK protein levels, while AAV-ARIH2 treatment significantly reduced viral load, underscoring its significant translational implications. In vivo, intranasal delivery of AAV-ARIH2 reduced lung viral load and inflammation, thereby reducing IAV-induced morbidity and mortality. Conclusion: ARIH2 emerges as a novel host cellular protein exploited by IAV to inhibit BIK degradation, thereby promoting viral replication. Enhancing ARIH2-mediated BIK ubiquitination presents a promising novel therapeutic strategy to reduce IAV replication and lung inflammation.

Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics. Most pandemics occurred upon adaptation of avian IAVs to humans. This adaptation includes a hallmark receptor-binding specificity switch of hemagglutinin (HA) from avian-type α2,3- to human-type α2,6-linked sialic acids. Complementary changes of the receptor-destroying neuraminidase (NA) are considered to restore the precarious, but poorly described, HA-NA-receptor balance required for virus fitness. In comparison to the detailed functional description of adaptive mutations in HA, little is known about the functional consequences of mutations in NA in relation to their effect on the HA-NA balance and host tropism. An understudied feature of NA is the presence of a second sialic acid-binding site (2SBS) in avian IAVs and absence of a 2SBS in human IAVs, which affects NA catalytic activity. Here we demonstrate that mutation of the 2SBS of avian IAV H5N1 disturbs the HA-NA balance. Passaging of a 2SBS-negative H5N1 virus on MDCK cells selected for progeny with a restored HA-NA balance. These viruses obtained mutations in NA that restored a functional 2SBS and/or in HA that reduced binding of avian-type receptors. Importantly, a particular HA mutation also resulted in increased binding of human-type receptors. Phylogenetic analyses of avian IAVs show that also in the field, mutations in the 2SBS precede mutations in HA that reduce binding of avian-type receptors and increase binding of human-type receptors. Thus, 2SBS mutations in NA can drive acquisition of mutations in HA that not only restore the HA-NA balance, but may also confer increased zoonotic potential.